This proposal aims to understand the effects of EphA4/ephrin-B1 signaling on the proliferation of progenitor cells during cerebral cortical development. Proper cell division during corticogenesis is essential for the appropriate formation and precise function of the mature brain. Thus, it is important to understand how the forming cerebral cortex regulates cell division. I propose that intercellular communication via EphA4/ephrin- B1 modulates cell division in cortical proliferative zones. The proposed experiments will characterize the influences of the receptor, EphA4, from those of its ligand, ephrin-B1, on the proliferation of cortical progenitor cells. Furthermore, experiments will address the means by which Eph signaling affects the cell cycle, investigating the consequences of different levels of Eph-mediated signaling on cell proliferation. Finally, the roles of particular domains within each protein on cortical proliferation will be assessed so that the molecular strategies underlying Eph modulation of cell division will be apparent. This grant application has two specific aims: SA1: Dissociate the effects of forward and reverse signaling of EphA4/ephrin-B1 in cortical progenitor cells and SA 2: Determine the protein domains in EphA4 and ephrin-BI underlying specific effects on cortical proliferation. To these ends, SA1 will use in vivo siRNA knockdown of the EphA4 receptor levels in cortical progenitor cells via In Utero Electroporation of embryonic mouse cortex to assess progenitor cell proliferation. Importantly, different levels of knock-down will be produced in these studies in order to identify the consequences of changing the balance of Eph/ephrin signaling. In these studies, cell- cycle kinetics following specific perturbation will be investigated in order to follow a cell's proliferative strategy. To complement the in vivo paradigm used in SA1, an in vitro proliferation model, cultures of cortical progenitors, will be used in SA2 to characterize the roles of discrete protein domains in EphA4/ephrin-B1 signaling in cortical proliferation. In neurosphere cultures derived from EphA4 or ephrin-B1 mutant mice, EphA4 or ephrin-B1 constructs containing wild type or mutated protein domains, respectively, will be introduced and the characteristics of the fully or partially rescued progenitor cells will be assessed. In this way, the role of Eph signaling in cortical proliferation will be ascertained and the molecular mechanisms will be revealed. Understanding how intercellular signaling modulates cell division in the cerebral cortex may provide insight into developmental disorders that could result from improper cell division. In addition, understanding the control of cell generation in development is likely to provide insight into the treatment of cortical injury in maturity.